Irreversible electroporation to control bleeding

ABSTRACT

A method of stopping or controlling bleeding by the placement of electrodes into or near the vicinity of vessels is disclosed. Then the application of electrical pulses causing irreversible electroporation of vessel and blood cells throughout the entire area of current flow the bleeding is stopped or controlled. The electric pulses irreversibly permeate the cell membranes, thereby invoking cell death. The irreversibly permeabilized cells are left in situ and are removed by the body immune system. Through the use of irreversible electroporation bleeding can be stopped or controlled without inducing thermal damage.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.60/532,588, filed Dec. 24. 2003, which application is incorporatedherein by reference.

FIELD OF THE INVENTION

This invention resides in the fields of electroporation of tissue and totreatments whereby unwanted bleeding is stopped.

BACKGROUND OF THE INVENTION

In many medical procedures, such as the treatment of benign or malignanttumors, it is important to be able to ablate the undesirable tissue in acontrolled and focused way without affecting the surrounding desirabletissue. Over the years, a large number of minimally invasive methodshave been developed to selectively destroy specific areas of undesirabletissues as an alternative to resection surgery. There are a variety oftechniques with specific advantages and disadvantages, which areindicated and contraindicated for various applications. For example,cryosurgery is a low temperature minimally invasive technique in whichtissue is frozen on contact with a cryogen cooled probe inserted in theundesirable tissue (Rubinsky, B., ed. Cryosurgery. Annu. Rev. Biomed.Eng. Vol. 2. 2000. 157-187.). The area affected by low temperaturetherapies, such as cryosurgery, can be easily controlled throughimaging. However, the probes are large and difficult to use.Non-selective chemical ablation is a technique in which chemical agentssuch as ethanol are injected in the undesirable tissue to cause ablation(Shiina, S., et al., Percutaneous ethanol injection therapy forhepatocellular carcinoma: results in 146 patients. AJR, 1993. 160: p.1023-8). Non-selective chemical therapy is easy to apply. However, theaffected area cannot be controlled because of the local blood flow andtransport of the chemical species. Elevated temperatures are also usedto ablate tissue. Focused ultrasound is a high temperature non-invasivetechnique in which the tissue is heated to coagulation usinghigh-intensity ultrasound beams focused on the undesirable tissue (Lynn,J. G., et al., A new method for the generation of use of focusedultrasound in experimental biology. J. Gen Physiol., 1942. 26: p.179-93; Foster, R. S., et al., High-intensity focused ultrasound in thetreatment of prostatic disease. Eur. Urol., 1993.23: p. 44-7).Electrical currents are also commonly used to heat tissue.Radiofrequency ablation (RF) is a high temperature minimally invasivetechnique in which an active electrode is introduced in the undesirabletissue and a high frequency alternating current of up to 500 kHz is usedto heat the tissue to coagulation (Organ, L. W., Electrophysiologicalprinciples of radiofrequency lesion making. Appl. Neurophysiol., 1976.39: p. 69-76). In addition to RF heating traditional Joule heatingmethods with electrodes inserted in tissue and dc or ac currents arealso common, (Erez, A., Shitzer, A. (Controlled destruction andtemperature distribution in biological tissue subjected to monoactiveelectrocoagulation) J. Biomech. Eng. 1980:102(1):42-9). Interstitiallaser coagulation is a high temperature thermal technique in whichtumors are slowly heated to temperatures exceeding the threshold ofprotein denaturation using low power lasers delivered to the tumors byoptical fibers (Bown, S. G., Phototherapy of tumors. World. J. Surgery,1983. 7: p. 700-9). High temperature thermal therapies have theadvantage of ease of application. The disadvantage is the extent of thetreated area is difficult to control because blood circulation has astrong local effect on the temperature field that develops in thetissue. The armamentarium of surgery is enhanced by the availability ofthe large number of minimally invasive surgical techniques in existence,each with their own advantages and disadvantages and particularapplications. This document discloses another minimally invasivesurgical technique for tissue ablation, irreversible electroporation. Wewill describe the technique, evaluate its feasibility throughmathematical modeling and demonstrate the feasibility with in vivoexperimental studies.

Electroporation is defined as the phenomenon that makes cell membranespermeable by exposing them to certain electric pulses (Weaver, J. C. andY. A. Chizmadzhev, Theory of electroporation: a review. Bioelectrochem.Bioenerg., 1996. 41: p. 135-60). Electroporation pulses are defined asthose electrical pulses that through a specific combination ofamplitude, shape, time length and number of repeats produce no othersubstantial effect on biological cells than the permeabilization of thecell membrane. The range of electrical parameters that produceelectroporation is bounded by: a) parameters that have no substantialeffect on the cell and the cell membrane, b) parameters that causesubstantial thermal effects (Joule heating) and c) parameters thataffect the interior of the cell, e.g. the nucleus, without affecting thecell membrane. Joule heating, the thermal effect that electricalcurrents produce when applied to biological materials is known forcenturies. It was noted in the previous paragraph that electricalthermal effects which elevate temperatures to values that damage cellsare commonly used to ablate undesirable tissues. The pulse parametersthat produce thermal effects are longer and/or have higher amplitudesthan the electroporation pulses whose only substantial effect is topermeabilize the cell membrane.

There are a variety of methods to electrically produce thermal effectsthat ablate tissue. These include RF, electrode heating, and inductionheating. Electrical pulses that produce thermal effects are distinctlydifferent from the pulses which produce electroporation. The distinctioncan be recognizing through their effect on cells and their utility. Theeffect of the thermal electrical pulses is primarily on the temperatureof the biological material and their utility is in raising thetemperature to induce tissue ablation through thermal effects.

The effect of the electroporation parameters is primarily on the cellmembrane and their utility is in permeabilizing the cell membrane forvarious applications. Electrical parameters that only affect theinterior of the cell, without affecting the cell membrane were alsoidentified recently. They are normally referred to as “nanosecondpulses”. It has been shown that high amplitude, and short (substantiallyshorter than electroporation pulses—nanoseconds versus millisecond)length pulses can affect the interior of the cell and in particular thenucleus without affecting the membrane. Studies on nanosecond pulsesshow that they are “distinctly different than electroporation pulses”(Beebe S J. Fox P M. Rec L J. Somers K. Stark R H. Schoenbach K H.Nanosecond pulsed electric field (nsPEF) effects on cells and tissues:apoptosis induction and tumor growth inhibition. PPPS-2001 Pulsed PowerPlasma Science 2001. 28th IEEE International Conference on PlasmaScience and 13th IEEE International Pulsed Power Conference. Digest ofTechnical Papers (Cat. No.01CH37251). IEEE. Part vol.1, 2001, pp.211-15vol.1. Piscataway,N.J., USA. Several applications have been identifiedfor nano-second pulses. One of them is for tissue ablation through aneffect on the nucleus (Schoenbach, K. H., Beebe, S. J., Buescher, K. S.Method and apparatus for intracellular electro-manipulation U.S. PattentApplication Pub No. US 2002/0010491 A1, Jan 24, 2002). Another is toregulate genes in the cell interior, (Gunderson, M. A. et al. Method forintracellular modification within living cells using pulsed electricalfields—regulate gene transcription and entering intracellular U.S.Patent application 2003/0170898 A1, Sep. 11, 2003). Electrical pulsesthat produce intracellular effects are distinctly different from thepulses which produce electroporation. The distinction can be recognizingthrough their effect on cells and their utility. The effect of theintracellular electrical pulses is primarily on the intracellularcontents of the cell and their utility is in manipulating theintracellular contents for various uses—including ablation. The effectof the electroporation parameters is primarily on the cell membrane andtheir utility is in permeabilizing the cell membrane for variousapplications, which will be discussed in greater detail later.

Electroporation is known for over half a century. It was found that as afunction of the electrical parameters, electroporation pulses can havetwo different effects on the permeability of the cell membrane. Thepermeabilization of the membrane can be reversible or irreversible as afunction of the electrical parameters used. In reversibleelectroporation the cell membrane reseals a certain time after thepulses cease and the cell survives. In irreversible electroporation thecell membrane does not reseal and the cell lyses. A schematic diagramshowing the effect of electrical parameters on the cell membranepermeabilization (electroporation) and the separation between: noeffect, reversible electroporation and irreversible electroporation isshown in FIG. 1 (Dev, S. B., Rabussay, D. P., Widera, G., Hofmann, G.A., Medical applications of electroporation, IEEE Transactions of PlasmaScience, Vol28 No 1, Feb. 2000, pp 206-223) Dielectric breakdown of thecell membrane due to an induced electric field, irreversibleelectroporation, was first observed in the early 1970s (Neumann, E. andK. Rosenheck, Permeability changes induced by electric impulses invesicular membranes. J. Membrane Biol., 1972. 10: p.279-290; Crowley, J.M., Electrical breakdown of biomolecular lipid membranes as anelectromechanical instability. Biophysical Journal, 1973. 13: p.711-724; Zimmermann, U., J. Vienken, and G. Pilwat, Dielectric breakdownof cell membranes,. Biophysical Journal, 1974. 14(11): p. 881-899). Theability of the membrane to reseal, reversible electroporation, wasdiscovered separately during the late 1970s (Kinosita Jr, K. and T. Y.Tsong, Hemolysis of human erythrocytes by a transient electric field.Proc. Natl. Acad. Sci. USA, 1977. 74(5): p. 1923-1927; Baker, P. F. andD. E. Knight, Calcium-dependent exocytosis in bovine adrenal medullarycells with leaky plasma membranes. Nature, 1978. 276: p. 620-622;Gauger, B. and F. W. Bentrup, A Study of Dielectric Membrane Breakdownin the Fucus Egg,. J. Membrane Biol., 1979. 48(3): p. 249-264).

The mechanism of electroporation is not yet fully understood. It isthought that the electrical field changes the electrochemical potentialaround a cell membrane and induces instabilities in the polarized cellmembrane lipid bilayer. The unstable membrane then alters its shapeforming aqueous pathways that possibly are nano-scale pores through themembrane, hence the term “electroporation” (Chang, D. C., et al., Guideto Electroporation and Electrofusion. 1992, San Diego, Calif.: AcademicPress, Inc.). Mass transfer can now occur through these channels underelectrochemical control. Whatever the mechanism through which the cellmembrane becomes permeabilized, electroporation has become an importantmethod for enhanced mass transfer across the cell membrane.

The first important application of the cell membrane permeabilizingproperties of electroporation is due to Neumann (Neumann, E., et al.,Gene transfer into mouse lyoma cells by electroporation in high electricfields. J. EMBO, 1982. 1: p. 841-5). He has shown that by applyingreversible electroporation to cells it is possible to sufficientlypermeabilize the cell membrane so that genes, which are macromoleculesthat normally are too large to enter cells, can after electroporationenter the cell. Using reversible electroporation electrical parametersis crucial to the success of the procedure, since the goal of theprocedure is to have a viable cell that incorporates the gene.

Following this discovery electroporation became commonly used toreversible permeabilize the cell membrane for various applications inmedicine and biotechnology to introduce into cells or to extract fromcells chemical species that normally do not pass, or have difficultypassing across the cell membrane, from small molecules such asfluorescent dyes, drugs and radioactive tracers to high molecular weightmolecules such as antibodies, enzymes, nucleic acids, HMW dextrans andDNA. It is important to emphasize that in all these applicationselectroporation needs to be reversible since the outcome of the masstransport requires for the cells to be alive after the electroporation.

Following work on cells outside the body, reversible electroporationbegan to be used for permeabilization of cells in tissue. Heller, R., R.Gilbert, and M.J. Jaroszeski, Clinical applications ofelectrochemotherapy. Advanced drug delivery reviews, 1999. 35: p.119-129. Tissue electroporation is now becoming an increasingly popularminimally invasive surgical technique for introducing small drugs andmacromolecules into cells in specific areas of the body. This techniqueis accomplished by injecting drugs or macromolecules into the affectedarea and placing electrodes into or around the targeted tissue togenerate reversible permeabilizing electric field in the tissue, therebyintroducing the drugs or macromolecules into the cells of the affectedarea (Mir, L. M., Therapeutic perspectives of in vivo cellelectropermeabilization. Bioelectrochemistry, 2001. 53: p. 1-10).

The use of electroporation to ablate undesirable tissue was introducedby Okino and Mohri in 1987 and Mir et al. in 1991. They have recognizedthat there are drugs for treatment of cancer, such as bleomycin andcys-platinum, which are very effective in ablation of cancer cells buthave difficulties penetrating the cell membrane. Furthermore, some ofthese drugs, such as bleomycin, have the ability to selectively affectcancerous cells which reproduce without affecting normal cells that donot reproduce. Okino and Mori and Mir et al. separately discovered thatcombining the electric pulses with an impermeant anticancer drug greatlyenhanced the effectiveness of the treatment with that drug (Okino, M.and H. Mohri, Effects of a high-voltage electrical impulse and ananticancer drug on in vivo growing tumors. Japanese Journal of CancerResearch, 1987. 78(12): p. 1319-21; Mir, L. M., et al.,Electrochemotherapy potentiation of antitumour effect of bleomycin bylocal electric pulses. European Journal of Cancer, 1991.27: p. 68-72).Mir et al. soon followed with clinical trials that have shown promisingresults and coined the treatment electrochemotherapy (Mir, L. M., etal., Electrochemotherapy, a novel antitumor treatment:first clinicaltrial. C. R. Acad. Sci., 1991. Ser. III 313(613-8)).

Currently, the primary therapeutic in vivo applications ofelectroporation are antitumor electrochemotherapy (ECT), which combinesa cytotoxic nonpermeant drug with permeabilizing electric pulses andelectrogenetherapy (EGT) as a form of non-viral gene therapy, andtransdermal drug delivery (Mir, L. M., Therapeutic perspectives of invivo cell electropermeabilization. Bioelectrochemistry, 2001. 53: p.1-10). The studies on electrochemotherapy and electrogenetherapy havebeen recently summarized in several publications (Jaroszeski, M. J., etal., In vivo gene delivery by electroporation. Advanced applications ofelectrochemistry, 1999. 35: p. 131-137; Heller, R., R. Gilbert, and M.J. Jaroszeski, Clinical applications of electrochemotherapy. Advanceddrug delivery reviews, 1999. 35: p. 119-129; Mir, L. M., Therapeuticperspectives of in vivo cell electropermeabilization.Bioelectrochemistry, 2001. 53: p. 1-10; Davalos, R. V., Real TimeImaging for Molecular Medicine through electrical Impedance Tomographyof Electroporation, in Mechanical Engineering. 2002, University ofCalifornia at Berkeley: Berkeley. p. 237). A recent article summarizedthe results from clinical trials performed in five cancer researchcenters. Basal cell carcinoma (32), malignant melanoma (142),adenocarcinoma (30) and head and neck squamous cell carcinoma (87) weretreated for a total of 291 tumors (Mir, L. M., et al., Effectivetreatment of cutaneous and subcutaneous malignant tumours byelectrochemotherapy. British Journal of Cancer, 1998. 77(12): p.2336-2342).

Electrochemotherapy is a promising minimally invasive surgical techniqueto locally ablate tissue and treat tumors regardless of theirhistological type with minimal adverse side effects and a high responserate (Dev, S. B., et al., Medical Applications of Electroporation. IEEETransactions on Plasma Science, 2000. 28(1): p. 206-223; Heller, R., R.Gilbert, and M. J. Jaroszeski, Clinical applications ofelectrochemotherapy. Advanced drug delivery reviews, 1999. 35: p.119-129). Electrochemotherapy, which is performed through the insertionof electrodes into the undesirable tissue , the injection of cytotoxicdugs in the tissue and the application of reversible electroporationparameters, benefits from the ease of application of both hightemperature treatment therapies and non-selective chemical therapies andresults in outcomes comparable of both high temperature therapies andnon-selective chemical therapies.

In addition, because the cell membrane permeabilization electrical fieldis not affected by the local blood flow, the control over the extent ofthe affected tissue by this mode of ablation does not depend on theblood flow as in thermal and non-selective chemical therapies. Indesigning electroporation protocols for ablation of tissue with drugsthat are incorporated in the cell and function in the living cells itwas important to employ reversible electroporation; because the drugscan only function in a living cell. Therefore, in designing protocolsfor electrochemotherapy the emphasize was on avoiding irreversibleelectroporation. The focus of the entire field of electroporation forablation of tissue was on using reversible pulses, while avoidingirreversible electroporation pulses, that can cause the incorporation ofselective drugs in undesirable tissue to selectively destroy malignantcells. Electrochemotherapy which employs reversible electroporation incombination with drugs, is beneficial due to its selectivity however, adisadvantage is that by its nature, it requires the combination ofchemical agents with an electrical field and it depends on thesuccessful incorporation of the chemical agent inside the cell.

An important concern in the studies of electrochemotherapy andelectrogenetherapy in living tissue is the effect of electroporation onblood flow. Martin et al., have found that when reversibleelectroporation is used for introducing genes into cells on the bloodvessel wall the blood vessels remain intact and their response tostimuli where indistinguishable from those of control vessels (Martin,J. B., Young, J. L., Benoit, J. N., Dean, D. A., Gene transfer to intactMesenteric arteries by electroporation, Journal of vascular research,2000, Vol 37:372-380). Ivanusa et al have found using MRI that withcertain electroporation pulses, which appear to be in the irreversibleelectroporation range, that the electroporation transiently butsignificantly reduced tumor blood flow (Ivanusa, T., Beravs, K.,Cemazar, M., Jevtic, V., Demsar, F., Sersa, G. MRI macromolecularcontrast agents as indicators of changed tumor blood flow, Radiol.Oncol. 2001; 35(2): 139-47). These findings are very different fromthose described here.

Sersa et al performed studies whose goal was to determine the effect ofelectrochemotherapy, reversible elctroporation with bleomycin orcisplatin, on tumor blood flow (Sersa, G., Sentjurc, M., Ivanusa, T.,Beravs, K., Kotnik, V., Coer, A., Swartz, H. M, Cemazar, M. Reducedblood flow and axygenation in SA-1 tumours after electrochemotherapywith cisplatin, Br. J. Cancer, 2002: 87(9):1047-54) (Sersa, G., Cemazar,M., Miklavcic, D. Tumor blood flow modifying effects ofelectrochemotherapy: a potential targeted mechanism radiol. Oncol 2003:37(1): 43-8). In the first of the papers they report reduced blood flowthat persisted for several days when using reversible electroporationwith cisplatin. In the second paper they report complete shut down ofblood flow after 24 hours when using reversible electroporation withbleomycin and 50% reduction in blood flow when using reversibleelectroporation with cisplatin.

The present inventors have found through histological analysis ofelectroporated tissue that in regions of tissue which are irreversibleelectroporated the local blood flow completely ceases, whereas outsidethat area the blood flow is not affected. This has the effect of localblood flow cessation and the appearance of global blood flow reduction.The applications of local blood flow cessation with irreversibleelectroporation are disclosed and described here.

Irreversible electroporation has been considered detrimental toconventional electrochemotherapy. However, the present invention showsthat it can be used to disrupt blood flow. Once blood flow to an area isstopped the cells in that area die. Thus, the method can be used invarious treatments as described here.

SUMMARY OF THE INVENTION

The present invention comprises a method whereby blood flow and suchbleeding from a vessel is stopped with the application of electricalpulses causing irreversible electroporation of cells making up vesselsand blood involved in unwanted bleeding. The electric pulsesirreversibly permeate the membranes, thereby invoking cell death. Thelength of time of the electrical pulses, the voltage applied and theresulting membrane permeability are all controlled within definedranges. The irreversibly permeabilized cells may be left in situ and maybe removed by natural processes such as the body's own immune system.Through the use of irreversible electroporation bleeding may becompletely disrupted or controlled without inducing thermal damage.

This concept of irreversible electroporation to control bleeding isdifferent from other forms of electrical therapies and treatments.Irreversible electroporation is different from intracellularelectro-manipulation which does not cause irreversible cell membranedamage. Further, irreversible electroporation is not electricallyinduced thermal coagulation, but rather a more benign method to destroyonly the targeted cells of vessels and blood involved in undesiredbleeding. The electroporation pulse should not have considerable thermaleffects, but irreversible break cell membranes of targeted cells andresult in cell death.

An electrical pulse can either have no effect on the cell membrane,effect internal cell components, reversibly open the cell membrane afterwhich cells can survive, or irreversibly open the cell membrane, afterwhich the cells die. Of these effects, irreversible electroporation isgenerally undesirable due to the possibility of instantaneous necrosisof the entire tissue affected by the electrical field, regardless of itsdiseased or healthy state. Irreversible electroporation is detrimentalin certain applications, such as gene therapy or electrochemotherapy,where the sole purpose of the electric pulses is to facilitate theintroduction of the drug or gene into the cells of a tissue withoutkilling the cell (Mir., L. M. and S. Orlowski, The basis ofelectrochemotherapy, in Electrochemotherapy, electrogenetherapy, andtransdermal drug delivery: Electrically mediated delivery of moleculesto cells, M. J. Jaroszeski, R. Heller, R. Gilbert, Editors, 2000, HumanaPress, p. 99-118).

In contrast, irreversible electroporation solely uses electrical pulsesto serve as the active means for tissue destruction by a specific means,i.e. by fatally disrupting the cell membrane. Electrochemotherapy may beselective, but it does require the combination of chemical agents withthe electrical field. Irreversible electroporation, althoughnon-selective to a degree, may be used to control undesirable bleedingas a minimally invasive surgical procedure with or without the use ofadjuvant drugs. Its non-selective mode of tissue ablation is acceptablein the field of minimally invasive surgery and is more comparable tocryosurgery, non-selective chemical ablation and high temperaturethermal ablation.

An aspect of the invention is a method whereby cells of vessels andblood are irreversibly electroporated by applying pulses of veryprecisely determined length and voltage. This may be done whilemeasuring and/or observing changes in electrical impedance in real timeand noting decreases at the onset of electroporation and adjusting thecurrent in real time to obtain irreversible cellular damage. Inembodiments where voltage is applied, the monitoring of the impedanceaffords the user knowledge of the presence or absence of pores. Thismeasurement shows the progress of the pore formation and indicateswhether irreversible pore formation, leading to cell death, hasoccurred.

An aspect of this invention is that the onset and extent ofelectroporation of cells in tissue can be correlated to changes in theelectrical impedance (which term is used herein to mean the voltage overcurrent) of the tissue. At a given point, the electroporation becomesirreversible. A decrease in the resistivity of a group of biologicalcells occurs when membranes of the cells become permeable due to poreformation. By monitoring the impedance of the biological cells in atissue such as vessels and blood therein, one can detect the averagepoint in time in which pore formation of the cells occurs, as well asthe relative degree of cell membrane permeability due to the poreformation. By gradually increasing voltage and testing cells in a giventissue one can determine a point where irreversible electroporationoccurs. This information can then be used to establish that, on average,the cells of the tissue have, in fact, undergone irreversibleelectroporation. This information can also be used to control theelectroporation process by governing the selection of the voltagemagnitude.

The invention provides the simultaneous irreversible electroporation ofmultitudes of cells providing a direct indication of the actualoccurrence of electroporation and an indication of the degree ofelectroporation averaged over the multitude. The discovery is likewiseuseful in the irreversible electroporation of biological tissue (massesof biological cells with contiguous membranes) for the same reasons. Thebenefits of this process include a high level of control over thebeginning point of irreversible electroporation.

A feature of the invention is that the magnitude of electrical currentduring electroporation of the tissue becomes dependent on the degree ofelectroporation so that current and pulse length are adjusted within arange predetermined to obtain irreversible electroporation of targetedcells of the tissue while minimizing cellular damage to surroundingcells and tissue.

An aspect of the invention is that pulse length and current areprecisely adjusted within ranges to provide more than mere intracellularelecctro-manipulation which results in cell death and less than thatwhich would cause thermal damages to the surrounding tissues.

Another aspect of the invention is that the electroporation is carriedout without adding drugs, DNA, or other materials of any sort to bebrought into the cells.

Another feature of the invention is that measuring current (in realtime) through a circuit gives a measurement of the average overalldegree of electroporation obtained.

Another aspect of the invention is that the precise electricalresistance of the tissue is calculated from cross-time voltagemeasurement with probe electrodes and cross-current measurement with thecircuit attached to electroporation electrodes.

Another aspect of the invention is that the precise electricalresistance of the tissue is calculated from cross-time voltagemeasurement with probe electrodes and cross-current measurement with thecircuit attached to electroporation electrodes.

Another aspect of the invention is that electrical measurements of thetissue can be used to map the electroporation distribution of thetissue.

These and further features, advantages and objects of the invention willbe better understood from the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures:

FIG. 1. is a graph showing a schematic relationship between fieldstrength and pulselength applicable to the electroporation of cells.

FIGS. 2A, 2B and 2C are each images of irreversibly electroporated areasfor two-electrode configurations using 10 mm center-to-center spacing asfollowing for FIGS. 2A, B and C: (2A) 0.5 mm (857V); (2B) 1.0 mm(1295V); (2C) 1.5 mm (1575V) diameter electrodes with a 680V/cmthreshold for irreversible electroporation.

FIGS. 3A, 3B, and 3C are images showing irreversibly electroporatedregions using a 680 V/cm threshold for a two-electrode confirmation withlmm diameter and 876V and 5 mm spacing for FIG. 3A; 1116V and 7.5 mm forFIG. 3B; and 1295V and 10 mm spacing for FIG. 3C.

FIGS. 4A, 4B and 4C are images showing the effect of electrode diameterfor a 4-electrode configuration with 10 mm spacing wherein FIG. 4A isfor 0.5 mm diameter and 940V; FIG. 4B is for 1.0 mm diameter and 1404Vand FIG. 4C is for 1.5 mm and 1685V.

FIGS. 5A, 5B and 5C are images showing the effect of electrode spacingfor a 4-electrode configuration wherein the electrode is 1 mm indiameter and FIG. 5A shows results with a 5 mm and 910V; FIG. 5B 7.5 mmand 1175V and FIG. 5C 10 mm and 1404V.

FIG. 6 is an image showing the irreversible (1295V, 680V/cm threshold)as compared to the reversible region (1300V, 360V/cm threshold) usingvirtually the same electrical parameters. 1300V is the most commonvoltage applied across two electrodes for ECT. The most common voltageparameters are eight 100 μs pulses at a frequency of 1 Hz. Applying asingle 800 μs pulse provides a conservative estimate of the heatingassociated with a procedure. The one second space normally betweenpulses will enlarge an area amount of heat to be dissipated through thetissue.

FIG. 7 is an image showing reversible electroporation with 1 mmelectrodes, 10 mm spacing. A voltage of 189V applied between theelectrodes induces reversible electroporation without any irreversibleelectroporation by not surpassing the 680V/cm irreversibleelectroporation threshold anyone in the domain. The shaded area isgreater than 360 V/cm.

FIGS. 8A and 8B show a comparison of the effect of blood flow andmetabolism on the amount of irreversible electroporation. FIG. 8A noblood flow or metabolism. FIG. 8B w_(b)=1 kg/m³, C_(b)=3640 J/(kg K),T_(b)=37° C., and q′″=33.8 kW/m³.

FIG. 9 is a schematic view of a liver between two cylindrical Ag/AgClelectrodes. The distance between the electrodes was 4 mm and the radiusof the electrodes is 10 mm. The electrodes were clamped with special rigparallel and concentric to each other. The liver lobe was compressedbetween the electrodes to achieve good contact.

FIG. 10 is a photo of a view of a liver which was electroporated byirreversible electroporation with two cylindrical surface electrodes of10 mm in diameter. Histology shows that the dark area is necrotic.

FIG. 11 is a photo of a cross section through an electroporated liver.Histology shows that the dark area is necrotic. The distance between thetwo A1 plates that hold the liver is exactly 4 mm. The electroporationelectrodes were 10 mm in diameter and centered in the middle of thelesion.

FIG. 12 shows the liver of calculated temperature distribution (C),upper panel , and electrical potential gradient (electroporationgradient) (V/cm), lower panel, for the in vivo experiment. The FIG. 12also shows conditions through a cross section of a liver slab throughthe center of the electroporated area. Height of the slab is 4 mm.

FIG. 13 combines FIGS. 11 and 12 to show a comparison between the extentof tissue necrosis (dark area) and the temperature and voltage gradientdistribution in the electroporated tissue. It is evident that most ofthe dark area was at a temperature of about 42 C following the 40milliseconds electroporation pulse. The edge of the dark area seems tocorrespond to the 300 V/cm electroporation gradient line.

FIG. 14 is a photo of a micrograph of the interface between irreversibleelectroporated liver and normal liver. The left hand side shows normalhepatocytes with clear nucleus and nuclei, well defined cell membraneand clean (flushed) sinusoids. The right hand side shows condensednuclei, no evidence of cell membrane, expanded cell border with noevidence of sinusoids. Disintegrated red blood cells are in what couldhave been the spaces of the sinusoids. No effect of flushing.

DETAILED DESCRIPTION OF THE INVENTION

Before the present methods, treatments and devices are described, it isto be understood that this invention is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit, unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.The present disclosure is controlling to the extent it conflicts withany incorporated publication.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “apulse” includes a plurality of such pulses and reference to “the sample”includes reference to one or more samples and equivalents thereof knownto those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DEFINITIONS

The term “reversible electroporation” encompasses permeabilization ofthe cell membrane through the application of electrical pulses acrossthe cell. In “reversible electroporation” the permeabilization of thecell membrane ceases after the application of the pulse and the cellmembrane permeability reverts to normal. The cell survives “reversibleelectroporation.” It is used as a means for introducing chemicals, DNA,or other materials into cells.

The term “irreversible electroporation” also encompasses thepermeabilization of the cell membrane through the application ofelectrical pulses across the cell. However, in “irreversibleelectroporation” the permeabilization of the cell membrane does notcease after the application of the pulse and the cell membranepermeability does not revert to normal. The cell does not survive“irreversible electroporation” and the cell death is caused by thedisruption of the cell membrane and not merely by internal perturbationof cellular components. Openings in the cell membrane are created and/orexpanded in size resulting in a fatal disruption in the normalcontrolled flow of material across the cell membrane. The cell membraneis highly specialized in its ability to regulate what leaves and entersthe cell. Irreversible electroporation destroys that ability to regulatein a manner such that the cell can not compensate and as such the celldies.

Invention in General

The invention provides a method and a system for destruction (ablation)of undesirable tissue. It involves the insertion (bringing)electroporation electrodes to the vicinity of the undesirable tissue andin good electrical contact with the tissue and the application ofelectrical pulses that cause irreversible electroporation of the cellsthroughout the entire area of the undesirable tissue. The cells whosemembrane was irreversible permeabilized are left in situ (not removed)to be removed by the body's immune system. Cell death is produced byinducing the electrical parameters of irreversible electroporation inthe undesirable area.

Electroporation protocols involve the generation of electrical fields intissue and are affected by the Joule heating of the electrical pulses.When designing tissue electroporation protocols it is important todetermine the appropriate electrical parameters that will maximizetissue permeabilization without inducing deleterious thermal effects. Ithas been shown that substantial volumes of tissue can be electroporatedwith reversible electroporation without inducing damaging thermaleffects to cells and has quantified these volumes (Davalos, R. V., B.Rubinsky, and L. M. Mir, Theoretical analysis of the thermal effectsduring in vivo tissue electroporation. Bioelectrochemistry, 2003. Vol61(1-2): p. 99-107). The electrical pulses required to induceirreversible electroporation in tissue are larger in magnitude andduration from the electrical pulses required for reversibleelectroporation. Further, the duration and strength of the pulsesrequired for irreversible electroporation are different from othermethodologies such as for intracellular electro-manipulation. Themethods are very different even when the intracellularelectro-manipulation is irreversible and is used to cause cell death,e.g. ablate the tissue of a tumor.

Typical values for pulse length for irreversible electroporation are ina range of from about 50 microseconds to about 2,000 microseconds orabout 75 microseconds to about 200 microseconds or about 100microseconds ±10 microseconds. This is significantly longer than thepulse length generally used in intracellular electro-manipulation whichis 1 microsecond or less—see published U.S. application 2002/0010491published Jan. 24, 2002.

The pulse is at voltage of about 110 v to 2,200 v ot 220 v to 550 v orabout 440 v±10% for irreversible electroporation. This is substantiallylower than that used for intracellular electro-manipulation which isabout 10,000 v/cm, see U.S. application 2002/0010491 published Jan. 24,2002.

The voltage expressed above is the voltage per cm. The electrodes may bedifferent shapes and sizes and be positioned at different distances fromeach other. The shape may be circular, oval, square, rectangular orirregular etc. The distance of one electrode to another may be 0.5 to 10cm., 1 to 5 cm., or 2-3 cm. The electrode may have a surface area of0.1-5 sq. cm. or 1-2 sq. cm.

The size, shape and distances of the electrodes can vary and such canchange the voltage and pulse duration used. Those skilled in the artwill adjust the parameters in accordance with this disclosure to obtainthe desired degree of electroporation and avoid thermal damage tosurrounding cells.

When using irreversible electroporation for tissue ablation, there maybe concern that the irreversible electroporation pulses will be as largeas to cause thermal damaging effects to the surrounding tissue and theextent of the tissue ablated by irreversible electroporation will not besignificant relative to that ablated by thermal effects. Under suchcircumstances irreversible electroporation could not be considered as aneffective tissue ablation modality as it will act in superposition withthermal ablation.

The present invention evaluates, through a mathematical model, themaximal extent of tissue ablation that could be accomplished byirreversible electroporation prior to the onset of thermal effects. Themodels focused on electroporation of liver tissue with two and fourneedle electrodes using available expiermental data. The liver waschosen because it is considered a potential candidate for irreversibleelectroporation ablation since in other tissues, such as skin and musclethe larger electrical pulses could cause or attenuate muscle and nerveexcitation. The results show that the area that can be ablated byirreversible electroporation prior to the onset of thermal effects iscomparable to that which can be ablated by electrochemotherapy,validating the use of irreversible electroporation as a potentialminimally invasive surgical modality.

Earlier studies have shown that the extent of electroporation can beimaged in real time with electrical impedance tomography (Davalos, R.V., B. Rubinsky, and D. M. Otten, A feasibility study for electricalimpedance tomography as a means to monitor tissue electroporation formolecular medicine. IEEE Transactions on Biomedical Engineering, 2002.49(4): p. 400-403). Irreversible electroporation, therefore, has theadvantage of a tissue ablation technique that is as easy to apply ashigh temperature ablation, without the need for adjuvant chemicals andwith real- time control of the affected area with electrical impedancetomography.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1

The mathematical model provided here shows that irreversible tissueablation can affect substantial volumes of tissue, without inducingdamaging thermal effects. To this end, the present invention uses theLaplace equation to calculate the electrical potential distribution intissue during typical electroporation pulses and a modified Pennes(bioheat), (Pennes, H. H., Analysis of tissue and arterial blood flowtemperatures in the resting forearm. J of Appl. Physiology., 1948. 1: p.93-122), equation to calculate the resulting temperature distribution.It is important to note that there are several forms of the bioheatequation which have been reviewed (Carney, C. K., Mathematical models ofbioheat transfer, in Bioengineering heat transfer, Y. I. Choi, Editor.1992, Academic Press, Inc: Boston. p. 19-152; Eto, T. K. and B.Rubinsky, Bioheat transfer, in Introduction to bioengineering, S. A.Berger, W. Goldsmith, and E. R. Lewis, Editors. 1996, Oxford Press).While the Pennes equation is controversial, it is nevertheless commonlyused because it can provide an estimate of the various biological heattransfer parameters, such as blood flow and metabolism. The modifiedPennes equation in this study contains the Joule heating term in tissueas an additional heat source.

The electrical potential associated with an electroporation pulse isdetermined by solving the Laplace equation for the potentialdistribution:∇·(σ∇φ)=0  (1)

-   -   where φ is the electrical potential and σ is the electrical        conductivity. The electrical boundary condition of the tissue        that is in contact with the leftmost electrode(s) on which the        electroporation pulse is applied is:        φ=V₀  (2)

The electrical boundary condition at the interface of the rightmostelectrode(s) is:φ=0  (3)

The boundaries where the analyzed domain is not in contact with anelectrode are treated as electrically insulative to provide an upperlimit to the electrical field near the electroporation electrodes and anupper limit to the temperature distribution that results fromelectroporation: $\begin{matrix}{\frac{\partial\phi}{\partial n} = 0} & (4)\end{matrix}$

Solving the Laplace equation enables one to calculate the associatedJoule heating, the heat generation rate per unit volume from anelectrical field (p):p=σ|∇φ| ²  (5)

This term is added to the original Pennes equation, (Pennes, H. H.,Analysis of tissue and arterial blood flow temperatures in the restingforearm. J of Appl. Physiology., 1948. 1: p. 93-122) to represent theheat generated from the electroporation procedure: $\begin{matrix}{{{\nabla{\cdot \left( {k\quad\Delta\quad T} \right)}} + {w_{b}\quad{c_{b}\left( {T_{a} - T} \right)}} + q^{m} + p} = {\rho\quad c_{p}\frac{\partial T}{\partial t}}} & (6)\end{matrix}$

To solve equation (4) it is assumed that the entire tissue is initiallyat the physiological temperature of 37° C.:T(x,y,z,0)=37  (7)

The outer surface of the analyzed domain and the surfaces of theelectrodes are taken to be adiabatic, which should produce an upperlimit to the calculated temperature distribution in the tissue:$\begin{matrix}{{\frac{\partial T}{\partial n} = 0}\quad{{on}\quad{the}\quad{electrodes}\quad{boundary}\quad{and}\quad{the}\quad{outer}\quad{surface}\quad{domain}}} & (8)\end{matrix}$

The analysis modeled conditions typical to tissue electroporation in theliver. The liver was chosen because it is the organ that most minimallyinvasive ablation techniques treat since cancer in the liver can beresolved by extirpation of the diseased area while surgical resection isnot possible in many cases for this organ (Onik, G., B. Rubinsky, and etal., Ultrasound-Guided Hepatic Cryosurgery in the Treatment ofMetastatic Colon Carcinoma. Cancer, 1991. 67(4): p. 901-907). Theelectroporation parameters, i.e. pulse parameters for reversible andirreversible electroporation where obtained from rat liver data(Miklavcic, D., et al., A validated model of in vivo electric fielddistribution in tissues for electrochemotherapy and for DNAelectrotransfer for gene therapy. Biochimica et Biophysica Acta, 2000.1523(1): p. 73-83; Suzuki, T., et al., Direct gene transfer into ratliver cells by in vivo electroporation. FEBS Letters, 1998. 425(3): p.436-440), but biological parameters corresponding to the human liverwere used in the analysis. Tissue thermal properties are taken fromreference (Duck, F. A., Physical Properties of Tissues: A ComprehensiveReference Book. 1990, San Diego: Academic Press) and the electricalproperties from reference (Boone, K., D. Barber, and B. Brown,Review—Imaging with electricity: report of the European Concerted Actionon Impedance Tomography. J. Med. Eng. Technol., 1997.21: p. 201-232) andare listed in table 1. The tissue is assumed isotropic andmacroscopically homogeneous.The intent of the analysis was to determinethe extent of the region in which reversible or irreversibleelectroporation is induced in the liver for various electroporationvoltages and durations while the maximal temperature in the tissue isbelow 50° C. Thermal damage is a time-dependent process described by anArhenius type equation (Henriques, F. C. and A. R. Moritz, Studies inthermal injuries: the predictability and the significance of thermallyinduced rate processes leading to irreversible epidermal damage. ArchPathol., 1947. 43: p. 489-502; Diller, K. R., Modeling of bioheattransfer processes at high and low temperatures, in Bioengineering heattransfer, Y. I. Choi, Editor. 1992, Academic Press, Inc: Boston. p.157-357),Ω=∫ξe ^(−E) ^(a) ^(/RT) dt  (9)

Where Ω is a measure of thermal damage, ξ is the frequency factor, E_(a)is the activation energy and R is the universal gas constant. A detaileddescription on the various degrees of thermal damage as described inEquation (9) above can be found in (Diller, K. R., Modeling of bioheattransfer processes at high and low temperatures, in Bioengineering heattransfer, Y. I. Choi, Editor. 1992, Academic Press, Inc: Boston. p.157-357).

A careful examination shows that the thermal damage is a complexfunction of time, temperature and all the parameters in Equation (9)above and that there are various degrees of thermal damage. In variousapplications or for various considerations it is possible to designirreversible electroporation protocols that induce some degree ofthermal damage, either in part of the electroporated region or at areduced level throughout the electroporated region. However, in thisexample we have chosen 50° C. as the target temperature for severalreasons. Thermal damage begins at temperatures higher than 42° C., butonly for prolonged exposures. Damage is relatively low until 50° C. to60° C. at which the rate of damage dramatically increases (Diller, K.R., Modeling of bioheat transfer processes at high and low temperatures,in Bioengineering heat transfer, Y. I. Choi, Editor. 1992, AcademicPress, Inc: Boston. p. 157-357). Therefore 50 C will be a relatively lowbound on the possible thermal effects during irreversibleelectroporation. It is anticipated that the electrical parameters chosenfor irreversible electroporation without a thermal effect could besubstantially longer and higher than those obtained from an evaluationfor 50 C in this example. Furthermore, since the Laplace and bioheatequations are linear, the results provided here can be extrapolated andconsidered indicative of the overall thermal behavior.

The analyzed configurations have two needles or four needle electrodesembedded in a square model of the liver. Needle electrodes are commonlyused in tissue electroporation and will be most likely also used in theliver (Somiari, S., et al., Theory and in vivo application ofelectroporative gene delivery. Molecular Therapy, 2000. 2(3): p.178-187). The square model of the liver was chosen large enough to avoidouter surface boundary effects and to produce an upper limit for thetemperature, which develops during electroporation in the liver. Foreach configuration the surface of one electrode is assumed to have aprescribed voltage with the other electrode set to ground. The effect ofthe spacing between the electrodes was investigated by comparingdistances of 5, 7.5 and 10 mm, which are typical. The electrodes werealso modeled with typical dimensions of 0.5, 1 and 1.5 mm in diameter.The blood flow perfusion rate was taken to zero or 1.0 kg/M³ s (Deng, Z.S. and J. Liu, Blood perfusion-based model for characterizing thetemperature fluctuations in living tissue. Phys A STAT Mech Appl, 2001.300: p. 521-530). The metabolic heat was taken to be either zero or 33.8kW/m³ (Deng, Z. S. and J. Liu, Blood perfusion-based model forcharacterizing the temperature fluctuations in living tissue. Phys ASTAT Mech Appl, 2001. 300: p. 521-530).

The calculations were made for an electroporation pulse of 800 μs. Thispulse duration was chosen because typically, reversible electroporationis done with eight separate 100 μs pulses, (Miklavcic, D., et al., Avalidated model of in vivo electric field distribution in tissues forelectrochemotherapy and for DNA electrotransfer for gene therapy.Biochimica et Biophysica Acta, 2000. 1523(1): p. 73-83) and thereforethe value we chose is an upper limit of the thermal effect in a pulsetime frame comparable to that of reversible electroporation.Consequently, the results obtained here are the lower limit in possiblelesion size during irreversible electroporation. It should be emphasizedthat we believe irreversible electroporation tissue ablation can be donewith shorter pulses than 800 μs. To evaluate the thermal effect, wegradually increased in our mathematical model the applied pulseamplitude for the 800 μs pulse length until our calculations indicatedthat the electroporation probe temperature reached 50° C., which weconsidered to be the thermal damage limit. Then, we evaluated theelectric field distribution throughout the liver.

A transmembrane potential on the order of 1V is required to induceirreversible electroporation. This value is dependent on a variety ofconditions such as tissue type, cell size and other external conditionsand pulse parameters. The primary electrical parameter affecting thetransmembrane potential for a specific tissue type is the amplitude ofthe electric field to which the tissue is exposed. The electric fieldthresholds used in estimating the extent of the region that wasirreversibly electroporated were taken from the fundamental studies ofMiklavcic, Mir and their colleagues performed with rabbit liver tissue(Miklavcic, D., et al., A validated model of in vivo electric fielddistribution in tissues for electrochemotherapy and for DNAelectrotransfer for gene therapy. Biochimica et Biophysica Acta, 2000.1523(1): p. 73-83). In this study, that correlated electroporationexperiments with mathematical modeling, they have found that theelectric field for reversible electroporation is 362 +/−21 V/cm and is637 +/−43 V/cm for irreversible electroporation for rat liver tissue.Therefore, in the analysis an electric field of 360 V/cm is taken torepresent the delineation between no electroporation and reversibleelectroporation and 680 V/cm to represent the delineation betweenreversible and irreversible electroporation.

All calculations were performed using MATLAB's finite element solver,Femlab v2.2 (The MathWorks, Inc. Natick, Mass.). To ensure mesh qualityand validity of solution, the mesh was refined until there was less thana 0.5% difference in solution between refinements. The baseline meshwith two 1 mm electrodes, 10 mm spacing had 4035 nodes and 7856triangles. The simulations were conducted on a Dell Optiplex GX240 with512 MB of RAM operating on Microsoft Windows 2000.

Results and Discussion

FIGS. 2 and 3 examine the effect of the electrode size and spacing onthe ablated area in a two-needle electroporation configuration. Inobtaining these figures, we ignored the effect of the blood flow andmetabolism in the heat transfer equation, which should give an upperlimit for the estimated ablation area. FIG. 2 compares the extent of theirreversible electroporated area for electroporation electrode sizes of0.5, 1 and 1.5 mm in diameter and a distance between electrodes of 10mm. The strong effect of the electrode size is evident. It is seen thatfor the smaller electrodes, the irreversibly electroporated area is notcontiguous, while for a 1.5 mm electrode the area of potential tissue,ablation has an elliptical shape with dimensions of about 15 mm by 10mm. In the brackets, we give the electroporation voltage for which theprobe temperature reaches 50° C. in these three configurations. It isseen that the range is from 857V for the 0.5 mm probe to 1575V for the1.5 mm probe. This is within the typical range of tissue electroporationpulses. FIG. 3 evaluates the effect of the spacing between theelectrodes. It is observed that in the tested range, the small dimensionof the contiguous elliptical shape of the ablated lesion remains thesame, while the larger dimension seems to scale with the distancebetween the electrodes.

FIGS. 2 and 3 demonstrate that the extent of tissue ablation withirreversible electroporation is comparable to that of other typicalminimally invasive methods for tissue ablation, such as cryosurgery(Onik, G. M., B. Rubinsky, and et. al., Ultrasound-guided hepaticcryosurgery in the treatment of metastatic colon carcinoma. Cancer,1991. 67(4): p. 901-907; Onik, G. M., et al., Transrectalultrasound-guided percutaneous radical cryosurgical ablation of theprostate. Cancer, 1993. 72(4): p. 1291-99). It also shows that varyingelectrode size and spacing can control lesion size and shape. The shapeand size of the ablated lesion can be also controlled by varying thenumber of electrodes used. This is shown in FIGS. 4 and 5, for afour-electrode configuration. These figures also compare the effect ofprobe size and spacing and the results were also obtained by ignoringthe effect of blood flow and metabolism in the energy equation. Again,it is seen that larger electrodes have a substantial effect on theextent of the ablated region and that the extent of ablation scales withthe spacing between the electrodes.

A comparison between reversible and irreversible electroporationprotocols can be achieved from FIGS. 6 and 7. In FIG. 6, an 800 μs, 1295V pulse was applied between two 1.5 mm diameter electrodes placed 10 mmapart. This produces a tissue temperature lower than 50° C. The figureplots the margin of the irreversibly electroporated region, i.e. the 680V/cm voltage-to-distance gradients and that of the reversibleelectroporated region, the 360 V/cm gradients. FIG. 7 was obtained fortwo 1 mm electrodes placed 10 mm apart. In this figure, we produced anelectroporated region that was only reversibly electroporated, i.e. withelectric fields lower than 360 V/cm. In comparing FIGS. 6 and 7, it isobvious that the extent of the ablated area possible throughelectrochemotherapy alone is substantially smaller than that throughirreversible electroporation alone.

The effect of blood flow and metabolism on the extent of irreversibleelectroporation is illustrated in FIG. 8. The figures compare asituation with metabolism and a relatively high blood flow rate to asituation without blood flow or metabolism. It is obvious thatmetabolism and blood perfusion have a negligible effect on the possibleextent of irreversible tissue electroporation. This is because theeffect of the Joule heating produced by the electroporation current issubstantially larger than the effects of blood flow or metabolism.

An even more conservative estimate for the thermal damage can beobtained by assuming that the tissue reaches 50° C. instantaneously,during the electroporation pulses such that the damage is defined asΩ=t _(p) ξe ^(−ΔE/RT)  (10)

Several values taken from the literature for activation energy andfrequency factor were applied to equation (10) with the pulse lengthscalculated in the examples above. Because the application of the pulseis so short, the damage would be near zero, many times less than thevalue (Ω=0.53) to induce a first degree burn (Diller, K. R., Modeling ofbioheat transfer processes at high and low temperatures, inBioengineering heat transfer, Y. I. Choi, Editor. 1992, Academic Press,Inc: Boston. p. 157-357). regardless of the values used for activationenergy and frequency factor.

Currently, tissue ablation by electroporation is produced through theuse of cytotoxic drugs injected in tissue combined with reversibleelectroporation, a procedure known as electrochemotherapy. The presentinvention shows that irreversible electroporation by itself producessubstantial tissue ablation for the destruction of undesirable tissuesin the body. The concern was that higher voltages required forirreversible electroporation would cause Joule heating and would inducethermal tissue damage to a degree that would make irreversibleelectroporation a marginal effect in tissue ablation. Using amathematical model for calculating the electrical potential andtemperature field in tissue during electroporation, the presentinvention shows that the area ablated by irreversible tissueelectroporation prior to the onset of thermal effects is substantial andcomparable to that of other tissue ablation techniques such ascryosurgery. Our earlier studies have shown that the extent ofelectroporation can be imaged in real time with electrical impedancetomography (Davalos, R. V., B. Rubinsky, and D. M. Otten, A feasibilitystudy for electrical impedance tomography as a means to monitor tissueelectroporation for molecular medicine. IEEE Transactions on BiomedicalEngineering, 2002. 49(4): p. 400-403; Davalos, R. V., et al., Electricalimpedance tomography for imaging tissue electroporation. IEEETransactions on Biomedical Engineering, 2004). Irreversibleelectroporation, therefore, has the advantage of being a tissue ablationtechnique, which is as easy to apply as high temperature ablation,without the need for adjuvant chemicals as required in electrochemicalablation and electrochemotherapy. In addition, a unique aspect ofirreversible electroporation is that the affected area can be controlledin real time with electrical impedance tomography.

Example 2

This example was developed to produce a correlation betweenelectroporation pulses and thermal effects. The system analyzed is aninfinitesimally small control volume of tissue exposed to anelectroporation voltage gradient of V (Volts/cm).The entire electricalenergy is dissipated as heat and there is no conduction of heat from thesystem. The calculations produce the increase in temperature with timeduring the application of the pulse and the results are a safe lowerlimit for how long a certain electroporation pulse can be administereduntil a certain temperature is reached. To generate the correlation anenergy balance is made on a control volume between the Joule heatingproduced from the dissipation of heat of the V (volt/cm) electricalpotential dissipating through tissue with an electrical conductivity ofσ ( ohm-cm) and the raise in temperature of the control volume made oftissue with a density ρ (g/cc) and specific heat, c, (J/g K). thecalculation produces the following equation for the raise in temperature(T) per unit time (t) as a function of the voltage gradients and thethermal and electrical properties of the liver. $\begin{matrix}{\frac{\mathbb{d}T}{\mathbb{d}t} = \frac{V^{2}\sigma}{\rho\quad c}} & \left( {2\text{-}1} \right)\end{matrix}$

The table below was obtained for the liver with the followingproperties:

-   -   Electrical resistivity of liver—8.33 Ohm-meter    -   Specific heat of liver—J/g K    -   Density of liver—1 g/cc

We obtain the following table: TABLE 1 Voltage Time per degree time from37 C. to Gradient - V (V/cm) C. rise (ms) 65 C. (ms) 50 1199.52 33586.56100 299.88 8396.64 150 133.28 3731.84 200 74.97 2099.16 250 47.981343.46 300 33.32 932.96 350 24.48 685.44 400 18.74 524.79 450 14.81414.65 500 12.00 335.87 550 9.91 277.57 600 8.33 233.24 650 7.10 198.74700 6.12 171.36 750 5.33 149.27 800 4.69 131.20 850 4.15 116.22 900 3.70103.66 950 3.32 93.04 1000 3.00 83.97 1050 2.72 76.16 1100 2.48 69.391150 2.27 63.49 1200 2.08 58.31 1250 1.92 53.74 1300 1.77 49.68 13501.65 46.07 1400 1.53 42.84 1450 1.43 39.94 1500 1.33 37.32

The second column of Table 1 gives the amount of time it takes for thetemperature of the liver to raise 1 C, when the tissue experiences theelectroporation pulse in column 1. The time for even a relatively highelectroporation voltage of 1500V/cm is of the order of 1.33 millisecondfor 1 C rise and 37.32 millisecond until a temperature of 65 C isreached. Using the equation (2-1) or Table 1 it is possible to evaluatethe amount of time a certain pulse can be applied without inducingthermal effects. Considering the typical electroporation parametersreported so far there is no limitation in the electroporation lengthfrom thermal considerations. Column 3 of Table 1 shows the time requiredto reach 65 C, which is where thermal damage may begin. The calculationsin this example give a lower limit for the extent of time in which acertain thermal effects will be induced by electroporation pulses. Formore precise calculations it is possible to use the equation developedin this example with equation (9) or (10) from Example 1.

Example 3

The goal of this experiment was to verify the ability of irreversibleelectroporation pulses to produce substantial tissue ablation in thenon-thermal regime. To this end we have performed experiments on theliver of Spraque-Dawley male rats (250 g to 350 g) under an approvedanimal use and care protocol. After the animals were anesthetized byinjection of Nembutal Sodium Solution (50 mg/ml Pentobarbital) the liverwas exposed via a midline incisions and one lobed clamped between twocylindrical electrodes of Ag/AgCl, with a diameter of 10 mm (In VivoMetric, Healdsburg, Calif.). The electrodes had their flat surfaceparallel; they were concentric and the liver between the electrodes wascompressed so that the lobes were separated by 4 mm. A schematic of theelectrodes and the liver is shown in FIG. 9. The liver was exposed to asingle electroporation pulse of 40 milliseconds. One electrode was setto 400 V and the other grounded. The rest of the liver was not incontact with any media and therefore is considered electricallyinsulated. After electroporation the rat was maintained under controlledanesthesia for three hours. Following exsanguination the liver wasflushed with physiological saline under pressure and fixed by perfusionwith formaldehyde. The liver was resected through the center of theelectroporated region and analyzed by histology. FIGS. 10 and 11 showthe appearance of the liver. Histology has determined that the dark areacorresponds to the region of tissue necrosis. The electrical field inthe electroporated liver and the temperature distribution werecalculated using the equations in Example 1, subject to one electrode ata voltage of 400V and the other grounded, for 40 milliseconds. The liverwas modeled as an infinite slab of 4 mm thickness, with concentriccylindrical electrodes (see FIG. 9). The results are shown in FIG. 12.FIG. 12 shows lines of constant voltage gradients (V/cm) and lines ofconstant temperature. It is evident that in the majority of theelectroporated tissue the temperature is about 42 C immediately afterthe pulse. The highest temperature occurs near the edge of thecylindrical electrodes, where it is about 50 C. FIG. 13 was obtained bybringing together FIGS. 11 and 12. Superimposing the calculated resultson the histological measurements reveals that the dark (necrotic) areamargin corresponds to electroporation parameters of about 300 V/cm. Theresults demonstrate that irreversible electroporation can inducesubstantial tissue necrosis without the need for chemical additives asin electrochemotherapy and without a thermal effect.

The results obtained by disrupting blood flow to a given area aredramatically shown in FIG. 14 which is a photo of a micrograph. Thismicrograph is from the interface between irreversible electroporatedliver and normal liver. The left hand side shows normal hepatocytes withclear nucleus and nuclei. The photo shows well defined cell membranesand clean (flushed) sinusoids. The right hand side of FIG. 14 showscondensed nuclei, no evidence of cell membrane, expanded cell borderwith no evidence of sinusoids. The disintegrated red blood cells shownin FIG. 14 are in what could have been the spaces of the sinusoids.Flushing is not believed to have had an effect on the results obtainedon the right hand side of FIG. 14.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

1. A method of controlling bleeding, comprising the steps of: (a)identifying a blood vessel undergoing bleeding; (b) placing a firstelectrode and a second electrode such that the identified blood vesselis positioned between the first and second electrodes; and (c) applyingelectrical pulses between the first and second electrodes in an amountsufficient to control bleeding from the vessel.
 2. The method of claim1, further comprising: (d) monitoring blood flow through the vessel; and(e) adjusting a characteristic of the electrical pulses to controlbleeding and minimize damage to surrounding tissue.
 3. The method ofclaim 1, wherein the electrical pulses are applied for a duration in arange of from about 5 microseconds to about 62 seconds.
 4. The method ofclaim 1, wherein the electrical pulses are applied for a period of about100 microseconds, i about 10 microseconds.
 5. The method of claim 1,wherein from about 1 to about 15 pulses are applied.
 6. The method ofclaim 1, wherein about eight pulses of about 100 microseconds each induration are applied.
 7. The method of claim 1, wherein the pulsesproduce a voltage gradient in a range of from about 50 volt/cm to about8000 volt/cm.
 8. The method of claim 1, wherein the first electrode isplaced at about 5 mm to 10 cm from the second electrode.
 9. The methodof claim 1, wherein the first and second electrodes are positioned in anarea of blood vessels upstream of a point where bleeding is occurring.10. The method of claim 2, further comprising: infusing a material intothe blood prior to applying the electrical pulses.
 11. The method ofclaim 10, wherein the material is an imaging agent.
 12. The method ofclaim 11, wherein the imaging agent is used to monitor blood flow. 13.The method of claim 1, wherein the first electrode and second electrodeare circular in shape.
 14. The method of claim 1, wherein the firstelectrode and the second electrode each have a surface area of about 1square centimeter.
 15. The method of claim 1, further comprising:monitoring blood flow in the blood vessels.
 16. The method of claim 15,further comprising: adjusting the electrical pulses based on real timeinformation of the monitored blood flow.
 17. The method of claim 1,further comprising: monitoring temperature of the identified tissue andadjusting the electrical pulses to maintain the temperature at 100° C.or less.
 18. The method of claim 17, wherein the temperature ismaintained at 50° C. or less.
 19. A method of claim 1, furthercomprising: adjusting applied voltage, length of pulses, and number ofpulses to completely stop bleeding and to minimize damage to tissue nearthe vessel.
 20. The method of claim 1, wherein: adjusting duration ofthe applied voltage is in accordance with the current-to-voltage ratioto control bleeding.
 21. The method of claim 20, wherein thecurrent-to-voltage ratio is adjusted based on temperature to maintainedtarget tissue temperature at 100° C. or less.
 22. The method of claim20, wherein the current-to-voltage ratio is adjusted based ontemperature to maintained target tissue temperature at 50° C. or less.23. The method of claim 15, further comprising: adjustingcurrent-to-voltage ratio based on monitored blood flow.
 24. A method ofcontrolling bleeding, comprising: (a) applying a voltage across a targetblood vessel; (b) continuously detecting the ratio of electric currentthrough the target vessel to voltage across the vessel as an indicationof controlling bleeding from the vessel; and (c) adjusting the appliedvoltage in accordance with changes in current-to-voltage ratio toachieve control of bleeding from the vessel.
 25. The method of claim 24,wherein: step (b) comprises continuously detecting thecurrent-to-voltage ratio as an indication of an onset of bleedingcontrol, and step (c) comprises adjusting duration of the appliedvoltage in accordance with the current-to-voltage ratio to achievecontrol of bleeding from the vessel.
 26. The method of claim 24,wherein: voltage is applied between two electrodes inserted in a mammal,and the electrodes are positioned to apply voltage across a targetedvessel; step (b) comprises further correlating the current-to-voltageratio with the temperature of the targeted vessel; and step (c)comprises adjusting the magnitude of the voltage while the targetedvessel is between the electrodes based on an averaged degree of bleedingcontrol from the targeted vessel.
 27. The method of claim 26, whereinthe current-to-voltage ratio is adjusted based on temperature tomaintain targeted vessel temperature at 60° C. or less.
 28. The methodof claim 27, wherein the current-to-voltage ratio is adjusted based ontemperature to maintain targeted vessel temperature at 50° C. or less.29. A device for controlling bleeding, comprising: first and secondelectrodes that position a bleeding blood vessel therebetween; a voltagegenerator means that applies a voltage between the first and secondelectrodes in a manner which provides a predetermined electric fieldaround the bleeding blood vessel for a time sufficient to controlbleeding from the vessel.
 30. The device of claim 29, wherein thegenerator means generates pulses of 100 microseconds ± about 10microseconds at a voltage gradient in a range of from about 50 volt/cmto about 8000 volt/cm.
 31. The device of claim 29, further comprising: ameans for adjusting the voltage and pulse duration of the generatormeans to control bleeding.
 32. The device of claim 29, furthercomprising: a means for monitoring blood flow.